Members of the genus Staphylococcus are major human pathogens, causing a wide variety of hospital and community acquired infections worldwide. The coagulase-positive species Staphylococcus aureus is well documented as a human opportunistic pathogen (Murray et al. Eds, 2003, Manual of Clinical Microbiology, 8th Ed., ASM Press, Washington, D.C.). Nosocomial infections caused by S. aureus are a major cause of morbidity and mortality. Some of the most common infections caused by S. aureus involve the skin, and they include furuncles or boils, cellulitis, impetigo, and postoperative wound infections at various sites. Some of the more serious infections produced by S. aureus are bacteremia, pneumonia, osteomyelitis, acute endocarditis, myocarditis, pericarditis, cerebritis, meningitis, scalded skin syndrome, and various abcesses. Food poisoning mediated by staphylococcal enterotoxins is another important syndrome associated with S. aureus. Toxic shock syndrome, a community-acquired disease, has also been attributed to infection or colonization with toxigenic S. aureus. 
Coagulase-negative Staphylococci had been regarded as harmless skin commensals prior to the 1970s, however, they are now recognized as important causes of human infections (Kloos, et al. (2004) Clin. Microbiol. Rev. 7:117-140). In addition to being among the most frequently isolated bacteria in clinical microbiology laboratories, coagulase-negative Staphylococci serve as reservoirs of antimicrobial resistance determinants (Bastos, et al. (1999) Eur. J. Clin. Microbiol. Infect. Dis. 18:393-398). As such, it is important to characterize and distinguish S. aureus strains from other, coagulase-negative Staphylococci.
S. aureus strains produce an extracellular thermostable nuclease (thermostable TNase) with a frequency similar to that at which they produce coagulase. The sequence of the gene encoding TNase, nuc, was first disclosed in 1985 (Kovacevi et al. (1985), J. Bact. 162:521-528). TNase is a 17 kDa protein that degrades both RNA and DNA at temperatures up to 100° C. TNase activity is not specific for S. aureus, however, S. aureus species-specific sequences exist. See, e.g., Brackstad, et al. (1992), J. Clin. Microbiol. 30:1654-1660; Zhang, et al. (2004), J. Clin. Microbiol. 42:4947-4955; Chesneau, et al. (1993) Mol. Cell. Probes 7:301-310, Wilson, et al. (1991) Appl. Environ. Microbiol. 57:1793-1798; Poulsen et al., (2003) J. Antimicrob. Chemo. 51:419-421, Costa et al., (2005), Diag. Microbiol. and Infect. Dis, 51: 13-17, Shittu et al., (2006), Diagn Microbiol Infect Dis. 2006 Jul. 17. To date, none of the S. aureus-specific nuc sequences have been proven to be clinically useful by way of a large specificity study. Therefore, there exists a need for oligonucleotides that have been proven to be both highly specific and sensitive, which are useful in rapid detection and identification of S. aureus from clinical samples.
Both S. aureus and coagulase-negative Staphylococci have a remarkable ability to accumulate additional antibiotic resistant determinants, resulting in the formation of multidrug-resistant strains. This resistance limits therapeutic options for treatment and substantially increases patient morbidity and mortality. Methicillin-resistant S. aureus (MRSA) emerged in the 1980s as a major clinical and epidemiologic problem in hospitals (Oliveira et al., (2002) Lancet Infect Dis. 2:180-189). MRSA are resistant to all β-lactams including penicillins, cephalosporins, carbapenems, and monobactams, which are the most commonly used antibiotics to cure S. aureus infections. MRSA infections can only be treated with more toxic and more costly antibiotics, which are normally used as the last line of defense. Since MRSA can spread easily from patient to patient via personnel, hospitals over the world are confronted with the problem to control MRSA.
Methicillin resistance in S. aureus is unique in that it is due to acquisition of DNA from other coagulase-negative staphylococci (CNS), coding for a surnumerary β-lactam-resistant penicillin-binding protein (PBP), which takes over the biosynthetic functions of the normal PBPs when the cell is exposed to β-lactam antibiotics. S. aureus normally contains four PBPs, of which PBPs 1, 2 and 3 are essential. The low-affinity PBP in MRSA, termed PBP 2a (or PBP2′), is encoded by the chromosomal mecA gene and functions as a β-lactam-resistant transpeptidase. The mecA gene is absent from methicillin-sensitive S. aureus but is widely distributed among other species of staphylococci and is highly conserved (Ubukata et al., (1990) Antimicrob. Agents Chemother. 34:170-172).
Nucleotide sequence determination of the DNA region surrounding the mecA gene from S. aureus strain N315 (isolated in Japan in 1982), led to the discovery that the mecA gene is carried by a novel genetic element, designated staphylococcal cassette chromosome mec (SCCmec), which is inserted into the chromosome. SCCmec is a mobile genetic element characterized by the presence of terminal inverted and direct repeats, a set of site-specific recombinase genes (ccrA and ccrB), and the mecA gene complex (Ito et al., (1999) Antimicrob. Agents Chemother. 43:1449-1458; Katayama et al., (2000) Antimicrob. Agents Chemother. 44:1549-1555). SCCmec is precisely excised from the chromosome of S. aureus strain N315 and integrates into a specific S. aureus chromosomal site in the same orientation through recombinases encoded by the ccrA and ccrB genes. Cloning and sequence analysis of the DNA surrounding the mecA gene from MRSA strains NCTC 10442 (the first MRSA strain isolated in England in 1961) and 85/2082 (a strain from New Zealand isolated in 1985) led to the discovery of two novel genetic elements that shared similar structural features of SCCmec. The three SCCmec have been designated type I (NCTC 10442), type II (N315) and type III (85/2082) based on the year of isolation of the strains (Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336). Hiramatsu et al. have found that the SCCmec DNAs are integrated at a specific site in the chromosome of methicillin-sensitive S. aureus (MSSA). The nucleotide sequence of the regions surrounding the left and right boundaries of SCCmec DNA (i.e. attL and attR, respectively), as well as those of the regions around the SCCmec DNA integration site (i.e. attBscc which is the bacterial chromosome attachment site for SCCmec DNA), were analyzed. Sequence analysis of the attL, attR and attBscc sites revealed that attBscc is located at the 3′ end of a novel open reading frame (ORF), orfX orfX encodes a putative 159-amino acid polypeptide that exhibits sequence homology with some previously identified polypeptides of unknown function (Ito et al., (1999) Antimicrob. Agents Chemother. 43:1449-1458). Two new types of SCCmec, designated type IV and type V were recently described (Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152, Ito et al., (2004) Antimicrob Agents Chemother. 48:2637-2651, Oliveira et al., (2001) Microb. Drug Resist. 7:349-360). Oliveira et al. also recently reported the existence of SCCmec type VI. Oliveira et al., (2006), Antimicrob Agents Chemother. 50:3457-3459. The sequence of the right extremity of some Staphylococcus strains classified as SCCmec type IV has been determined. See, Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152; Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336; Oliveira et al., (2001) Microb. Drug Resist. 7:349-360. Sequences from S. aureus strains CA05 and 8/6-3P, classified as SCCmec type IV, were nearly identical over 2000 nucleotides to that of type II SCCmec of S. aureus strain N315 (Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152; Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336).
Methods to detect and identify MRSA based on the detection of the mecA gene and S. aureus-specific chromosomal sequences have been described. See, Schuenck et al., Res. Microbiol., (2006), in press, Shittu et al., (2006), Diagn Microbiol Infect Dis. July 17, Grisold et al., (2006), Methods Mol. Biol. 345 : 79-89, Costa et al., (2005), Diag. Microbiol. and Infect. Dis, 51: 13-17, Mc Donald et al., (2005), J. Clin. Microbiol., 43: 6147-6149, Zhang et al., (2005), J. Clin. Microbiol. 43: 5026-5033, Hagen et al. (2005), Int J Med Microbiol. 295:77-86, Maes, et al. (2002) J. Clin. Microbiol. 40:1514-1517, Saito et al., (1995) J. Clin. Microbiol. 33:2498-2500; Ubukata et al., (1992) J. Clin. Microbiol. 30:1728-1733; Murakami et al., (1991) J. Clin. Microbiol. 29:2240-2244; Hiramatsu et al., (1992) Microbiol. Immunol. 36:445-453). Furthermore, Levi and Towner (2003), J. Clin. Microbiol., 41:3890-3892 and Poulsen et al. (2003), J Antimicrob Chemother., 51:419-421 describe detection of methicillin resistance in coagulase-negative Staphylococci and in S. aureus using the EVIGENE™ MRSA Detection kit.
However, because the mecA gene is widely distributed in both S. aureus and coagulase-negative staphylococci, each of the methods described above are incapable of discriminating between samples containing both methicillin-sensitive S. aureus (“MSSA”) and methicillin-resistant coagulase-negative staphylococci, and samples that contain only MRSA or that have both methicillin-sensitive S. aureus and MRSA.
To address this problem, Hiramatsu et al. developed a PCR-based assay specific for MRSA that utilizes primers that hybridize to the right extremities of DNA of SCCmec types I-III in combination with primers specific to the S. aureus chromosome, which corresponds to the nucleotide sequence on the right side of the SCCmec integration site. (U.S. Pat. No. 6,156,507, hereinafter the “'507 patent”). More recently, Zhang et al., (2005), J. Clin. Microbiol. 43: 5026-5033, described a multiplex assay for subtyping SCCmec types Ito V MRSA. Nucleotide sequences surrounding the SCCmec integration site in other staphylococcal species (e.g., S. epidermidis and S. haemolyticus) are different from those found in S. aureus, therefore multiplex PCR assays that utilize oligonucleotides that hybridize to the right extremities of SCCmec and the S. aureus chromosome have the advantage of being specific for the detection of MRSA.
The PCR assay described in the '507 patent also led to the development of “MREP typing” (mec right extremity polymorphism) of SCCmec DNA (Ito et al., (2001) Antimicrob. Agents Chemother. 45:1323-1336; Hiramatsu et al., (1996) J. Infect. Chemother. 2:117-129). The MREP typing method takes advantage of the fact that the nucleotide sequences of the three MREP types differ at the right extremity of SCCmec DNAs adjacent to the integration site among the three types of SCCmec. Compared to type I, type III has a unique nucleotide sequence while type II has an insertion of 102 nucleotides to the right terminus of SCCmec. The MREP typing method described by Hiramatsu et al. uses the following nomenclature: SCCmec type I is MREP type i, SCCmec type II is MREP type ii, and SCCmec type III is MREP type iii. Hiramatsu later revised this nomenclature in view of the publication of the sequences of the genomes of strains N315 and Mu50, since the sequences revealed that SCCmec elements are located downstream of orfX. Consequently, MREP can now be referred to as MLEP (mec left extremity polymorphism) (Chongtrakool et al., (2006), Antimicrob. Agents Chemother. 50:1001-1012).
Recently, Chongtrakool et al. proposed replacing the SCCmec nomenclature with new nomenclature. Chongtrakool et al., (2006), Antimicrob. Agents Chemother. 50:1001-1012. Chongtrakool et al.'s proposed nomenclature is based on the structure of SCCmec elements and has three features. The first feature is a description of the SCC type and is defined by ccr type and mec class. The second feature is the description of the J regions (junkyard regions), which are part of the SCCmec element, located between and around the mec and ccr complexes. The third feature is the enumeration which allows the numbering of ccr type and J regions according to their time of identification.
As stated above, SCCmec types II and IV have the same nucleotide sequence to the right extremity. Consequently, the MREP (or MLEP according to recent revision) typing method described above cannot differentiate the SCCmec type IV described by Hiramatsu et al. (Ma et al., (2002) Antimicrob. Agents Chemother. 46:1147-1152) from SCCmec type II).
We recently described DNA sequences and regions in MRSA named MREJ. PCT Application No. PCT/CA02/00824. The phrase MREJ refers to the mec right extremity junction mec right extremity junction. MREJ's are approximately 1 kilobase (kb) in length and include sequences from the SCCmec right extremity as well as bacterial chromosomal DNA to the right of the SCCmec integration site. Importantly, MREJ sequences provide advantages over MREP/MLEP sequences in classifying MRSA in that MREJ/MLEJ sequences enable the differentiation between strains classified as SCCmec type II and SCCmec type IV. As discussed in PCT Application No. PCT/CA02/00824, the strains that Hiramatsu classified as MREP types i-iii fall under MREJ types i-iii according to the MREJ typing system. We recently identified novel MREJ types iv-xx, and developed nucleic acid assays with improved ubiquity capable of detection and identification of MRSA of MREJ types i-xx. (Huletsky et al., 2004, J. Clin. Microbiol. 42:1875-1884, International Patent Application PCT/CA02/00824, U.S. patent application Ser. No. 11/248,438). Based on the revision of MREP to MLEP, one can understand that previously called MREJ types could now be reclassified as MLEJ (mec left extremity junction). The skilled artisan will appreciate that any S. aureus and MRSA classification system is contemplated in the methods disclosed herein, as sequences can specifically detect S. aureus and identify those which are resistant to methicillin.
Maes et al. describe a PCR assay to discriminate S. aureus from coagulase negative Staphylococci and to determine methicillin resistance in blood cultures (Maes, et al. (2002) J. Clin. Microbiol. 40:1514-1517). The assay described in Maes et al. cannot distinguish MRSA from methicillin-resistant coagulase-negative Staphylococci.
Poulsen et al. describe detection of methicillin resistance in coagulase-negative Staphylococci and in S. aureus using the EVIGENE™ MRSA Detection kit. The assay described in Poulsen et al. cannot discriminate between a sample that has both methicillin-sensitive S. aureus and methicillin-resistant coagulase-negative staphylococci, and a sample that contains only MRSA or that has both methicillin-sensitive S. aureus and MRSA.
Accordingly, there remains a need for a rapid assay to detect and identify both MRSA and methicillin-sensitive S. aureus in the same reaction and to be able to distinguish S. aureus from coagulase-negative Staphylococci in the same reaction.